The two signal model and the concept of costimulation are well engrained in our understanding of T cell regulation. It is well established that costimulation through CD28 can have a dramatic effect on T cell activation, survival, tolerance, and differentiation. However, in spite of considerable interest and effort, the regulation and mechanism of CD28 costimulation are still poorly understood. CD28 signaling is mediated through the recruitment of cytosolic signaling molecules to specific motifs within the cytosolic tail (CT) domain of CD28. In an effort to understand how ligand binding to the lumenal domain of CD28 could transduce changes to the cytosolic domains to initiate signaling, we established a FRET assay. CFP and YFP were fused to the end of the CT of CD28. CD28 is a disulfide-linked dimer and when CD28-CFP and CD28-YFP were co-expressed, high level of FRET was directed, indicating that the ends of the CT domains are in close proximity within the CD28 dimer. When CD28 was recruited to the immunological synapse, the level of FRET was reduced, indicating that there was some structural change in the orientation of the CT domains within the CD28 dimer. Surprisingly, this change in FRET was not mediated by CD28 ligand binding, but instead was mediated through TCR signaling, even in the absence of CD28 ligand binding. This suggests that TCR signaling can activate CD28, possibly impacting on ligand binding (in analogy to inside-out signaling for integrin activation), immunological synapse recruitment, or signal transduction. The overall goal of this R21 application is to identify the mechanism and functional consequence of this TCR- mediated change in the orientation of the CD28 CT domains. We will do this through two Specific Aims. 1, Determine the molecular events associated with TCR induced changes in the orientation of the CD28 CT domains. 2, Determine whether TCR signaling regulates CD28 ligand binding. PUBLIC HEALTH RELEVANCE: T cell activation requires the specific recognition of pathogen-specific proteins by the TCR and co-signaling through costimulatory molecules, most notably CD28. Although the function of CD28 is well described, the molecular events associated with CD28 function as not well understood. We have made the surprising finding that the TCR may modify the conformation of CD28 and so regulate the function of CD28.